Monoclonal antibodies against human protein Mcm3, process for their production, and their use

ABSTRACT

The present invention relates to monoclonal antibodies against the human Mcm3 protein, hybridoma cell lines, that produce such antibodies, procedures for the production and their use, pharmaceutical compositions comprising a monoclonal antibody according to the present invention, their use for the prevention and treating of certain diseases and well as methods relating to the prevention and treatment of diseases associated with Mcm3 expression. Monoclonal antibodies according to the invention detect and bind human Mcm3 monospecifically both in immunohistological and immunobiochemical detection systems. The process for the production of these monoclonal antibodies comprises an initial screening of excess hybridoma supernatant with immunobiochemical methods followed by a second screening of positive hybridoma by means of an immunohistochemical method.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/937,649, which is the U.S. National Phase of InternationalApplication No. PCT/EP00/02910, filed Mar. 31, 2000, designating theUnited States and claiming priority under 35 U.S.C. § 119 to GermanApplication No. DE 199 15 057.5, filed Apr. 1, 1999. The entireteachings of the above applications are incorporated herein byreference.

TECHNICAL FIELD

[0002] The present invention relates to monoclonal antibodies againsthuman protein Mcm3, processes for their production and their use.

PRIOR ART

[0003] Mcm proteins were first described in the barm S. cerevisiae. Itis known, that these proteins play an important role in the initiationof DNA replication, which was shown in the barm by its decisive role inthe transmittance of extra chromosome DNA segments, minichromosomes(Maine et al., Genetics, 1984, 106:365-385). This feature was the basisfor the naming for these proteins, minichromosome maintenance, Mcm. Theproteins of the Mcm family are highly conserved with respect toevolution.

[0004] At present six proteins (Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, Mcm7) aredescribed in the human system which with other cell cycle dependentstructures form a protein complex that is necessary for DNA replication,and which were already postulated as DNA replication licence factors byJ. J. Blow and R. A. Laskey in 1988 (Nature, 332:546-548). Mcm3 proteinplays an important role by forming a biochemical strong bond with Mcm5(A. Richter, R. Knippers, Eur. J. Biochem., 1997, 247:136-141). SinceMcm3 and the other members of the Mcm family have such a basic functionin the cell cycle, detection systems are desired, preferablyimmunobiochemical and immunohistological detections. Such detections areof need because new parameters for medical diagnosis, preferably incancer diagnosis, can be achieved therewith.

[0005] It is known that human Mcm protein is immunogenic in the rabbit(Thommes et al., Nucleic Acid Res., 1992, 20:1069-1074). But the knownpolyclonal antisera either do not react monospecifically inimmunobiochemical analyses (Western Blot) and/or are not applicablequickly and without problems in routine immunohistology (Hu, B., et al.,Nucleic Acid Res., 1993, 21: 5289-5293). Therefore, there is no tool athand that can serve as a detection method for Mcm3 in medical diagnosis.

SUMMARY OF THE INVENTION

[0006] The object of the present invention is therefore to provide meansthat detect quickly and monospecifically Mcm3 protein in biochemical andalso in histological systems conducted alone or together in combination.This detection can be conducted alone or in combination with other knownmarkers.

[0007] According to the invention this is achieved by a monoclonalantibody directed against Mcm3 protein and being applicable both inimmunobiochemical and in immunohistochemical detection systems, wherebythese detections can be conducted alone or in combination.

[0008] Further, hybridomas producing monoclonal antibodies according tothe invention are disclosed.

[0009] Another aspect of the invention is the provision of diagnosticcompositions and detection kits comprising the monoclonal antibodyaccording to the present invention.

[0010] Yet another aspect is the use of the monoclonal antibodyaccording to the present invention for the detection of Mcm3 in asample.

[0011] Moreover, processes are disclosed relating to the production of amonoclonal antibody and hybridoma, respectively, according to thepresent invention.

[0012] Finally, the present invention relates to pharmaceuticalpreparations and medicines containing the monoclonal antibody and theuse of the monoclonal antibody for the preparation of a medicament forthe treatment of certain diseases.

[0013] Also, within the scope of the invention are methods for treatingdiseases or disorders which are associated with an aberrant Mcm3 levelor activity or which can benefit from modulation of the activity orlevel of Mcm3. The methods comprise administering, e.g., either locallyor systemically to a subject, a pharmaceutically effective amount of acomposition comprising an Mcm3 antibody according to the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0014] According to the invention a monoclonal antibody directed againstMcm3 protein and being applicable both in immunobiochemical and inimmunohistochemical detection systems, whereby these detections can beconducted alone or in combination is provided.

[0015] The monoclonal antibody according to the present invention can beobtained from any animal or the human being, whereby the monoclonalantibodies of the mouse are preferred.

[0016] Further, the monoclonal antibody may be altered biochemically, bygenetic manipulation, or it may be synthetic, with the antibody possiblylacking portions completely or in parts, said portions being necessaryfor the recognition of Mcm3 and being substituted by others impartingfurther advantageous properties to the antibody.

[0017] A hybridoma cell line producing a preferred monoclonal antibodyof the present invention, namely, a monoclonal mouse antibody with saidabove-mentioned detection, was deposited at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b,D-38124, Braunschweig, Germany under Accession No. DSM ACC2388 on Feb.16, 1999.

[0018] The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) Mcm3. Examples of immunologicallyactive portions of immunoglobulin molecules include F(ab) and F(ab′)₂fragments which can be generated by treating the antibody with an enzymesuch as pepsin. The invention provides monoclonal antibodies that bindMcm3. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of Mcm3. Amonoclonal antibody composition thus typically displays a single bindingaffinity for Mcm3 with which it immunoreacts.

[0019] A disease, a disorder or condition “associated with” or“characterized by” an aberrant Mcm3 activity refers to a disease,disorder or condition in a subject which is caused by or contributed toby an aberrant Mcm3 activity.

[0020] The term “treating” as used herein is intended to encompasscuring as well as ameliorating at least one symptom of the condition ordisease.

[0021] Monoclonal anti-Mcm3 antibodies can be prepared by immunizing asuitable subject with an Mcm3 immunogen. An appropriate immunogenicpreparation can contain, for example, recombinantly expressed Mcm3protein or a chemically synthesized Mcm3 polypeptide. The preparationcan further include an adjuvant, such as Freund's complete or incompleteadjuvant, or similar immunostimulatory agents. Immunization of asuitable subject with an immunogenic Mcm3 preparation induces ananti-Mcm3 antibody response.

[0022] The anti-Mcm3 antibody titer in the immunized subject can bemonitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized Mcm3. If desired,the antibody molecules directed against Mcm3 can be isolated from themammal (e.g., from the blood) and further purified by well knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the antibodytiters are highest, antibody-producing cells can be obtained from thesubject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKoehler and Milstein (1975) Nature 256:495-497) (see also, Brown et al.(1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. US.4 76:2997-3 1;and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human Bcell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing monoclonal antibody hybridomas is well known(see generally R. H. Kenneth, in Monoclonal Antibodies: A new Dimensionin Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980);E. A. Lerner (1981) Yale J: Biol. Med., 54:387402; M. L. Gefter et al.(1977) Somatic Cell Genet. 3:23136). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with Mcm3 immunogen as described above, and theculture supernatants of the resulting hybridoma cells are screened toidentify a hybridoma producing a monoclonal antibody that binds Mcm3.

[0023] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatinganti-Mcm3 monoclonal antibodies (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med, cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Immortal cell lines are mouse myelomacell lines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS l/l-Ag4-1; P3×63-Ag8.653 or Sp2/O-Ag14myeloma lines. Typically, HAT-sensitive mouse myeloma cells are fused tomouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cellsresulting from the fusion are then selected using HAT medium, whichkills unfused myeloma cells (unfused splenocytes die after several daysbecause they are not transformed). Hybridoma cells producing amonoclonal antibody of the invention are detected by screening thehybridoma culture supernatants for antibodies that bind Mcm3, e.g.,using a standard ELISA assay.

[0024] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-Mcm3 antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with Mcm3 to thereby isolateimmunoglobulin library members that bind Mcm3. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP® Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol.Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram etal. (1992) Proc. Natl. Acad. Sci. USA 89:35763580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) J\tUC. Acid Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

[0025] Additionally, recombinant anti-Mcm3 antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,S67; Cabilly et al. European Patent Application 12S,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229: 1202-1207; Oi et al.(1986) Bio Techniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones etal. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Seidler et al. (1988) J. Immunol. 141:4053-4060. An anti-Mcm3monoclonal antibody can be used to isolate Mcm3 by standard techniques,such as affinity chromatography or immunoprecipitation. An anti-Mcm3antibody can be used to detect Mcm3 protein (e.g., in a cellular lysateor cell supernatant) in order to evaluate the abundance and pattern ofexpression of Mcm3. Anti-Mcm3 antibodies can be used diagnostically tomonitor protein levels in tissue as part of a clinical testingprocedure, e.g., to, for example, determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, (-galactosidase, oracetylcholinesterase); examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride, Cy-dyes, Alexa-dyes or phycoerythrin; an example of aluminescent material includes luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin, and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ; ³⁵S or ³H.

[0026] Preferably, monoclonal antibodies according to the invention canbe produced with the initial screening strategy described further below.Since a plurality of prepared hybridoma are either not monospecificallyagainst Mcm3 protein or are applicable only in immunobiochemicaldetection systems but not in immunohistological systems and vice versa,initial examination of generated hybridoma cells requires this strategyto produce monoclonal antibodies according to the invention which haveboth properties.

[0027] For the production of genetically altered and/or syntheticantibodies having the properties according to the invention one canstart e.g. from monoclonal antibodies obtained as described above. Forthis it is suitable to analyse the Mcm3 binding regions of themonoclonal antibodies and to identify the parts that are necessary andunnecessary for the detection described above. Then the necessaryportions can be modified and the unnecessary portions can be eliminatedcompletely or in part and can be substituted, respectively, by portionsimparting further advantageous properties to the antibodies. Also,portions not within the binding regions of the antibodies can bemodified, eliminated, or substituted. It is known by the skilled personthat particularly the DNA recombination technology is suitable for theabove measures.

[0028] Monoclonal antibodies according to the invention aredistinguished by detecting Mcm3 monospecifically both in biochemical andhistological detecting systems. The antibodies are therefore suitablefor the fast detection of a Mcm3 expression in very different samples.

[0029] Within the present context, the term “sample” is intended tocover all types of samples suitable for the purpose of the invention.Examples of such samples are serum, sputum, urine, liquor, tissue, andbiopsies. In particular the sample may be a blood sample or agynaecological sample.

[0030] Because of these features the antibodies according to theinvention are excellently suitable in the application of diagnosticproblems, in which comparatively the tissue topological distributionanalysis, e.g. determined by immunohistochemistry with quantitativeexpression parameters, e.g. obtained by Western Blot orimmunoprecipitation, shall be analysed.

[0031] With the antibodies according to the invention the monospecificdetection of the Mcm3 expression can be performed reliably in one-by-oneconducted immunobiochemical detection methods such as ELISA, WesternBlot, and immunoprecipitation, the Western Blot hereby being preferred,or immunohistochemical tissues, preferably on routine fixed and paraffinembedded tissue. For this the antibodies according to the invention maybe labelled, if it is appropriate, as described above, or employed incombination with labelled antibodies directed against them or otherreagents.

[0032] Monoclonal antibodies according to the invention can inhibit invivo the assembly of DNA precursors and therefore inhibit cellproliferation. Thus, these antibodies or the above mentioned derivativesof the same are suitable for the therapy of states of a disease whichare accompanied by raised cell proliferation. Examples of such diseasesare tumours, allergies, autoimmune diseases, scar formation,inflammations and rheumatic diseases as well as the suppression ofdefence reactions of transplantations.

[0033] For the production of pharmaceutical composition or a medicinethe monoclonal antibodies according to the invention can be used aloneor combined with common carriers, adjuvants and/or additives. Theantibodies are suitable for the systemic, local, subcutaneous,intrathecal and topical application and for application by enema. Forthis they can be applied solved in suitable solvents, preferably asaqueous solution, in the form of liposomes, as emulsion or in solidstate, e.g. as powder or in the form of microcapsules.

[0034] Alternatively, a monoclonal antibody of the present invention canbe administered in a combined method of treatment with a differentpharmaceutically active agent. Pharmaceutically active agents, that canbe formulated with the monoclonal antibodies of the present invention,or alternatively can be administered in a combined method of treatment,can be for instance antibodies, in particular monoclonal antibodies,against other antigens, thus providing a “cocktail” containing amonoclonal antibody of the present invention and one or more(monoclonal) antibodies against other antigens involved in thepathogenesis of the relevant disease state.

[0035] Further active agents, that can be formulated with the monoclonalantibodies of the present invention, or alternatively can beadministered in a combined method of treatment, especially in order toproduce a therapeutically useful effect, depend on the disease state tobe cured and are, for instance, commercially available gamma globulinand immune globulin products, antibiotics, antimicrobial products,antibacterial and antitumor agents or a mixture of two or more of them.

[0036] Monoclonal antibodies according to the invention can be employedin a particularly advantageous way in the therapy of tumours, namely assuch or in combination with other therapeuticals and forms of therapyrespectively, such as radiatio, that is resistant against conventionaltumour therapeuticals. Such resistances occur in unspecific cytostaticasuch as vinblastin or cisplatin either secondarily, i.e. after repeatedapplication, or exist primarily at certain tumours, such as carcinoma ofthe kidneys.

[0037] The dosages of such antibodies will vary with the condition beingtreated and the recipient of the treatment, but will be in the range of1 to about 100 mg for an adult patient preferably 1 to 10 mg usuallyadministered daily for a certain period. A two part dosing regime may bepreferable wherein 1 to 5 mg are administered.

[0038] In another preferred embodiment, the detection of Mcm3 isconducted in combination with the detection of the proteins Ki-67 andp27.

[0039] One of the most cited cell cycle associated proteins, used forhistopathologic diagnostics within the past 16 years, is the Ki-67protein (Scholzen T and Gerdes J (2000) J Cell Physiol 182: 311-322;Gerdes J, Schwab U, Lemke H and Stein H (1983). Int.J.Cancer 31, 13-20).The Ki-67 protein is expressed in proliferating cells, but rapidlydisappears when cells enter a resting state (Baisch, H, and Gerdes, J(1987). Cell Tissue Kinet. 20(4), 387-391). Clinical studies have shownthat the Ki-67 antigen is an independent prognostic marker in manydifferent human neoplasms, e.g. breast cancer (Jansen R L, Hupperets PS, Arends J W, Joosten-Achjanie S R, Volovics A, Schouten H C, andHillen H F (1998). Br. J. Cancer, 78: 460-465), soft tissue sarcoma,meningeomas (Perry A, Stafford S L, Scheithauer B W, Suman V J, andLohse C M (1998). Cancer 82: 2262-2269), prostate cancer (Mashal R D,Lester S, Corless C, Richie J P, Chandra R, Propert K J, and Dutta A(1996) Cancer Res 56(18):4159-63) and non-Hodgkin lymphoma (Gerdes etal. (1984), J. Immunol. 133: 1710-1715).

[0040] The protein p27 belongs to the family of cyclin dependent kinaseinhibitors (CDKI), which regulate cell cycle progression by binding andinactivating cyclin-dependent-kinases complexes at defined checkpointswithin the cell cycle (Toyoshima H, and Hunter T (1994) Cell78(1):67-74). The expression of p27 serves as a robust marker fordifferentiation in normal developing tissue and also in tumorsdisplaying deregulated growth (Lloyd R V, Jin L, Qian X, and Kulig E(1997) Am J Path 150: 401-407., Zhang P, Wong C, DePinho R A, Harper JW, and Elledge S J (1998) Genes Dev 12(20):3162-3167).

[0041] Performing combined staining of tissues detecting the threeproteins simultaneously, allow a more detailed assessment of cellproliferation and differentiation processes that determine individualtumor growth. Mcm3 protein is expressed in cells that have ceased toproliferate, but are not terminally differentiated according to theabsence of p27 protein expression, whereas Ki-67 is expressed inproliferating cells only. P27 can be found in quiescent cells but not inproliferating cells. Ki-67, Mcm3 and p27 provide one set of parameterswhich define complementary biological properties that are suitable for adetailed characterization of disordered cell growth and tumorgenesis.Tumor diagnostics may also benefit from a combined assessment of thesemarkers which may be of help to choose the most appropriate therapyconcept for an individual patient.

[0042] The present invention will be illustrated by the followingexamples:

EXAMPLES Example 1

[0043] Production of Monoclonal Antibodies According to the Invention.

[0044] Mice were used for immunisation. Recombinant human Mcm3 proteinwas used as antigen.

[0045] Record of Immunisation and Fusion

[0046] Day 1: 100 μg Mcm3 protein in 100 μl PBS (phosphate bufferedsaline) were mixed completely and thoroughly with 100 μl Freund'sadjuvant and were injected subsequently into a mouse.

[0047] Day 14: 50 μg Mcm3 protein in 100 μl PBS were mixed completelyand thoroughly with 100 μl Freund's adjuvant and were injectedsubsequently into a mouse.

[0048] Day 21: 50 μg Mcm3 protein in 100 μl PBS were mixed completelyand thoroughly with 100 μl Freund's adjuvant and were injectedsubsequently into a mouse.

[0049] Day 37: 50 μg Mcm3 protein in 100 μl PBS were mixed completelyand thoroughly with 100 μl Freund's adjuvant and were injectedsubsequently into a mouse.

[0050] On day 39 the mouse was killed painlessly. Spleen cells wereremoved and fused with myeloma cells. Hybridoma that reached full growthwere obtained

[0051] Screening of Hybridoma Supernatant and Cloning.

[0052] At first supernatants of the hybridoma which reached full growthwere tested in a spot-blot-assay. For this 1 ml recombinant human Mcm3in PBS (2 ng/ml) was placed on 1 cm×0.5 cm sized pieces ofnitrocellulose membrane. These pieces are placed in a 48 well-plate anddried for 15 minutes at room temperature. Subsequently incubation wasmade with blocking puffer (PBS, 0.005 Tween 20, 4% gelatine) for 45minutes at room temperature. After several washing steps with PBS (0.05%Tween 20, 0.5% gelatine) incubation was made with the hybridomasupernatant for 60 minutes at room temperature. After several washingsteps with PBS (0.05% Tween 20, 0.5% gelatine) a commercially availablephosphatase-coupled goat-anti-mouse antibody, Dianova, Hamburg (dilutionaccording to the instruction of the producer 1:10000) was added. Afterincubation for 1 hour at room temperature with PBS (0.05% Tween 20, 0.5%gelatine) the alkaline phosphatase detection reaction was conducted withthe developer solution (36 mM 5′ bromo-4-chloro-3-indolylphosphate; 400mM nitro blue tetrazolium, 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mMMgCl₂) for 10 minutes at room temperature.

[0053] The hybridoma supernatant which were tested positive in thespot-blot were tested subsequently immunohistologically. For thisparaffin sections, e.g. tonsil, were dehydrated according to standardprocedures (2×100% xylene, 2×100% EtOH, 2×70% EtOH, 2×40% EtOH),followed by washing briefly in water. The sections were then cooked incitrate buffer pH 6 (2.1 g citric acid monohydrate for 1l, adjust with2N NaOH to pH 6) in a pressure cooker for 1-5 minutes. After opening thecooker the sections were washed immediately in cold (RT) TBS, followedby incubation with hybridoma supernatant in a humid chamber for 30minutes. After washing in TBS for several times antibodies bound to thesections were detected by means of the indirect immunoperoxidase method,stained with hemalum, embedded and evaluated microscopically.

[0054] The antibody according to the present invention shows thefollowing staining pattern. The antibody predominantly reacts with thenuclei of cells in proliferative regions, indicated by the fact thatcells of the dark zone within the germinal centers of human tonsilsstain positive for Mcm3. Likewise cells near the basal layer of thenormal mucosa react with the Mcm3 specific antibody. It should be notedthat Mcm3 staining was also seen in the intermediate and upper layerwhich belong to the non-proliferating cell compartment of the oralmucosa.

[0055] Hybridoma which were positive both in the spot-blot and inimmunohistology, were cloned and recloned until they were monoclonal.Independent monoclonal antibodies were obtained. A hybridoma cell lineproducing a monoclonal antibody according to the invention was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Mascheroder Weg 1b, D-38124, Braunschweig, Germany underAccession No. DSM ACC2388 on Feb. 16, 1999.

Example 2

[0056] Western Blot Analysis of Cell Lysates with a Polyclonal RabbitAnti-Mcm3 Antibody and a Monoclonal Antibody According to the Invention.

[0057] Cell lysates of the cell line HELA (H) and CHO (C) were appliedto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE). The proteins separated in the gel were transferred to anitrocellulose membrane in a wet-blotting chamber over night. Thismembrane was then incubated with a diluted rabbit anti Mcm3 antiserum(Hu, B., et al., Nucleic Acid Res., 1993, 21: 5289-5293) (0.15 μg/ml)for 1 hour at room temperature. After several washing steps with PBS(0.05% Tween 20, 0.5% gelatin) a commercially availablephosphatase-coupled goat-rabbit antibody (dilution according to theinstructions of the producer, Dianova, Hamburg 1:10000) was added. Afterincubation of 1 hour at room temperature and once more washing inTNT-buffer (150 mM NaCl, 10 mM Tris ph 7.5, 0.05% Tween 20) thedetection was conducted with the chemiluminescence method with the ECLsystem (Amersham Life Science, Braunschweig) according to theinstructions of the producer.

[0058] It turned out that the polyclonal anti-Mcm3 rabbit antibodyrevealed, besides the expected prominent main protein band with anapparent molecular weight of 105 kDa, further proteins in the molecularweight range between 50 kDa and 90 kDa.

[0059] The monoclonal anti-Mcm3 antibody according to the inventionrevealed only the expected protein band with an apparent molecularweight of 105 kDa.

[0060] Thus, it is clearly demonstrated by Western Blot analysis thatthe antibody according to the present invention recognises only one bandwhile the polyclonal antibody detects further bands in the range of 90to 50 kDa.

[0061] Further, in immunohistochemical studies, the monoclonal antibodyaccording to the present invention demonstrates its usefulness for thedetection of Mcm3.

Example 3

[0062] Immunoprecipitation with Anti Mcm3 Antibodies According to theInvention

[0063] 1 μg anti-Mcm3 primary antibody is added to 10 μl Dyna-beads(Dynal M280 sheep-anti-mouse, Dynal, Hamburg) and incubated for 30minutes at 4° C. under rolling.

[0064] Cell preparations (1×10⁶ Cells) are taken up inimmunoprecipitation buffer (18 mM Tris/HCl, 150 mM NaCl, 0.3%hexadecylmethyl-ammoniumbromide, 5 mM EDTA and 1 mM DTT) comprisingprotease inhibitors, cooked for 5 minutes, cooled on ice and centrifuged(5 minutes, 14000 rpm). The excess liquid is added to the complex ofDynalbeads/primary antibodies and incubated at 4° C. for 30 minutes on aroller.

[0065] Then the tube is placed in a Dynal magnetic concentrator for 20seconds and the excess liquid is removed. Magnetic beads are resuspendedin 500 μl NET (Tris/HCl 18 mM, NaCl 150 mM, EDTA 5 mM, DTT 1 mM) placedagain in the magnetic concentrator and the supernatant is removed after20 seconds. In this way, the beads are washed several times.

[0066] The so purified Mcm3 can then be analysed by means of SDS-PAGE.

Example 4

[0067] Micro Injection of Anti-Mcm3 Antibodies According to theInvention in Nuclei of Permanent Cell Line Cells.

[0068] HEp-2 were cultivated on CELLocate® cover slips formicroinjection and used in the logarithmic growth period. Anti-Mcm3antibodies according to the invention and an irrelevant controlantibody, respectively, were microinjected with a transjector and micromanipulator into the nuclei under light microscopy control (pressure ofinjection 130 hPa; time of injection between 0,3 and 0,5 seconds). Theinjected cells were then cultivated with bromodesoxyuridin (BrdU)containing (0.1 mM) medium for 6 hours. After fixation (5 minutes 4%paraformaldehyde at room temperature) the cover slips were washed threetimes in Tris-buffered saline (TBS), incubated in 100% EtOH for 10minutes at −20° C. followed by permeation of the adhering cells bytransferring directly in 0.1% Triton X-100 TBS for 10 minutes by roomtemperature. The injected antibodies were then detected with acommercially available Cy3 coupled goat anti mouse antibody, Dianova,Hamburg (dilution according to the instructions of the producer inPBS/10% bovine serum albumin). The preparations were first incubated in2M HCl for 60 minutes at 37° C. for the detection of BrdU fixed into thecells. Subsequently, the preparations were washed first several timeswith distilled water, then two times with PBS. Then incubation with acommercially available FITC labelled anti BrdU antibody, BoehringerMannheim, Mannheim (dilution according to the instructions of theproducer) overnight at 4° C. in a humid chamber was performed. Then thepreparations were washed thoroughly five times in PBS for 10 minutes,covered with DABCO (1,4-diazabicyclo[2,2,2]octane)in 90% glycerol, andevaluated by fluorescence microscopy.

[0069] It turned out that almost all cells injected with controlantibodies incorporated also BrdU, hence passing during the experimentthe normal cell cycle. In contrast only between 20% to 50% of the cellsthat had been injected with anti-Mcm3 antibodies according to theinvention incorporated BrdU. The proliferation of these cells wastherefore inhibited by the antibodies according to the invention.

[0070] References

[0071] 1. Maine et al., Genetics, 1984, 106:365-385

[0072] 2. J. J. Blow and R. A. Laskey, Nature, 1988, 332:546-548

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What is claimed is:
 1. A method for detecting a proliferating cell, acancer cell or a neoplastic cell in a sample using a monoclonal antibodywhich binds specifically to human Mcm3, in which said monoclonalantibody reacts with the same epitope of human Mcm3 as the monoclonalantibody obtainable from the hybridoma cell line with deposit number DSMACC2388.
 2. A method according to claim 1, wherein said monoclonalantibody binds to human Mcm3 both immunohistologically andimmunobiochemically.
 3. A method according to claim 1, wherein saidmonoclonal antibody is selected from the group consisting of: arecombinant antibody, a chimeric antibody, a humanized antibody, asynthetic antibody and a genetically altered antibody.
 4. A methodaccording to claim 1, wherein said monoclonal antibody is labeled with adetectable substance.
 5. A method according to claim 4, wherein saiddetectable substance comprises a fluorescent, radioactive, enzymatic orluminescent label.
 6. A method according to claim 1, wherein saidmonoclonal antibody is produced by the hybridoma cell line with thedeposit number DSM ACC2388.
 7. A method according to claim 1, whereinthe proliferating, cancer or neoplastic cell is detectedimmunohistologically, immunocytologically or immunobiochemically.
 8. Amethod according to claim 1, wherein the sample is selected from thegroup consisting of: serum, a blood sample, sputum, urine, liquor, atissue, a biopsy, a gynaecological sample and a fine needle aspiration.9. A method according to claim 1, further comprising detecting Ki-67 andp27.
 10. A method according to claim 9, wherein Ki-67 and p27 aredetected by the use of antibodies thereto.
 11. A method of exposing acell to a monoclonal antibody which specifically binds to human Mcm3 fordisrupting or preventing the cell cycle of a cell, DNA replication by acell, or proliferation of a cell, in which the monoclonal antibodyreacts with the same epitope of human Mcm3 as the monoclonal antibodyobtainable from the hybridoma cell line with deposit number DSM ACC2388.12. A method according to claim 11, wherein said monoclonal antibody isselected from the group consisting of: a recombinant antibody, achimeric antibody, a humanized antibody, a synthetic antibody and agenetically altered antibody.
 13. A method according to claim 11,wherein said monoclonal antibody is produced by the hybridoma cell linewith the deposit number DSM ACC2388.
 14. A method for the treatment orprevention of an Mcm3 associated disease, the method comprisingadministering to an individual in need of such treatment atherapeutically effective amount of a monoclonal antibody whichspecifically binds to human Mcm3, in which the monoclonal antibodyreacts with the same epitope of human Mcm3 as the monoclonal antibodyobtainable from the hybridoma cell line with deposit number DSM ACC2388.15. A method according to claim 14, wherein said disease is selectedfrom the group consisting of: tumours, allergies, auto-immunopathies,scar formation, inflammation, rheumatic diseases and the suppression ofdefence reactions of transplantations.
 16. A method according to claim14, wherein said monoclonal antibody is selected from the groupconsisting of: a recombinant antibody, a chimeric antibody, a humanizedantibody, a synthetic antibody and a genetically altered antibody.
 17. Amethod according to claim 14, wherein said monoclonal antibody isproduced by the hybridoma cell line with the deposit number DSM ACC2388.18. A pharmaceutical composition comprising a monoclonal antibody whichspecifically binds to human Mcm3, wherein said monoclonal antibodyreacts with the same epitope of human Mcm3 as the monoclonal antibodyobtainable from the hybridoma cell line with deposit number DSM ACC2388,together with a pharmaceutically acceptable carrier, adjuvant oradditive.
 19. A method for the production of a hybridoma expressing amonoclonal antibody which specifically binds to human Mcm3, comprisingthe steps of: (a) immunizing an animal with human Mcm3; (b) obtaining alymphocyte from the animal immunized with human Mcm3; (c) immortalizingthe lymphocyte to produce a hybridoma; and (d) selecting a hybridomawhich expresses a monoclonal antibody which reacts with the same epitopeof human Mcm3 as the monoclonal antibody obtainable from the hybridomacell line with deposit number DSM ACC2388.
 20. A method according toclaim 19, in which a hybridoma is selected which expresses a monoclonalantibody which reacts both immunobiochemically and immunohistochemicallywith human Mcm3.
 21. A method for the production of a monoclonalantibody comprising expressing the antibody from a hybridoma obtained bya process according to claim
 19. 22. A method for the production of amonoclonal antibody comprising expressing the antibody from a hybridomaobtained by a process according to claim
 20. 23. A monoclonal antibodycapable of specific binding to human Mcm3 in which the monoclonalantibody reacts with the same epitope of human Mcm3 as the monoclonalantibody obtainable from the hybridoma cell line with deposit number DSMACC2388.
 24. A monoclonal antibody according to claim 23, wherein saidmonoclonal antibody is labeled with detectable substance.
 25. Amonoclonal antibody according to claim 24, wherein the detectablesubstance comprises a fluorescent, radioactive, enzymatic or aluminescent label.
 26. A monoclonal antibody according to claim 23,which is capable of binding to human Mcm3 both immunohistologically andimmunobiochemically.
 27. A monoclonal antibody according to claim 23,wherein said antibody is selected from the group consisting of: arecombinant antibody, a chimeric antibody, a humanized antibody, asynthetic antibody and a genetically altered antibody.
 28. A monoclonalantibody according to claim 23, wherein said antibody is produced by thehybridoma cell line with the deposit number DSM ACC2388.
 29. A hybridomacell line expressing a monoclonal antibody which binds to human Mcm3, inwhich the monoclonal antibody reacts with the same epitope of human Mcm3as the monoclonal antibody obtainable from the hybridoma cell line withdeposit number DSM ACC2388.